Earlier studies have led to the characterization of the protein components of the zona pellucida (ZP) from a variety of mammalian species, with the majority of the work being performed in the murine and porcine species. The reported number of discrete proteins comprising the ZP from different mammalian species varies (Dunbar et al, 1981; 1994; Timmons and Dunbar, 1988; Bleil and Wassarman, 1980). Murine studies have revealed that the ZP is composed of three sulfated glycoproteins, referred to as ZP1, ZP2 and ZP3. Extensive work in this model has resulted in the identification and isolation of the primary receptor for sperm located at the level of the zona pellucida, a glycoprotein called zona pellucida protein 3 (ZP3) (Bleil and Wassarman, 1980; Wassarman, 1990 a and b). The binding of sperm to ZP is supported by ZP3 and complementary binding protein(s) present in the sperm plasma membrane (Saling, 1989; Wassarman, 1990; Saling, 1991). Genes homologous to the ZP2 and/or ZP3 genes have been cloned for the mouse (ZP3 and ZP2), hamster (ZP3), human (ZP3), rabbit (rc75), and marmoset (Ringuette et al., 1986; Liang et al., 1990; Kinloch et al., 1990; Chamberlin & Dean, 1990; Liang & Dean, 1993; Lee et al., 1993; Thillai-Koothan et al., 1993). Genes encoding ZP2 and ZP3 are conserved among mammals and sequences of ZP3 cDNA coding regions show extensive homology between species studied so far.
Cloning cDNAs encoding ZP3 has made the expression of recombinant ZP3 in tissue culture cell lines possible and represents the potential to obtain large amounts of recombinant ZP3. The expression of biologically active recombinant ZP3 has been reported, at least, in the mouse (Kinloch et al, 1991; Beebe et al, 1992) and human (van Duin et al, 1994; Barratt et al, 1994; Burks et al, 1995). In the mouse, some of these recombinant proteins have demonstrated partial or full biological activity in ligand-receptor or acrosome reaction assays. Expression of recombinant ZP proteins is not restricted to those of the mouse and human species. Prasad et al (1996) demonstrated that recombinant rabbit 55 kDa protein (which is thought to be the rabbit homologue of mouse ZP1) purified from a baculovirus expression system could be used to generate a polyclonal antiserum which was then employed to study the localization of the native 55 kDa protein in rabbit zona.
On the other hand, recombinant human ZP3 has been expressed using several approaches, i.e., Escherichia coli (Chapman and Barratt, 1996), in vitro transcription and translation systems (Whitmarsh et al, 1996), Chinese hamster ovary (CHO) cells (van Duin et al, 1994; Barratt and Hornby, 1995; Brewis et al, 1996) and in African green monkey kidney (COS) cells (Burks et al, 1995). In the human, however, full biological activity, which includes the ability to bind spermatozoa in a specific fashion and to induce the acrosome reaction, has not been fully demonstrated for such products. This is possibly due, among other reasons, to inadequate or incomplete glycosylation of the recombinant protein (Chapman and Barratt, 1997).
In the human system, production of a pure recombinant ZP3 glycoprotein in a biologically active form has been fraught with technical difficulties. Expressing recombinant ZP3 protein with in vitro transcription and translation systems and in Escherichia coli has shown a variable acrosome reaction-inducing activity. However, no direct or specific sperm-binding ability using homologous sperm-ZP bioassays has been reported for such non-glycosylated products. In addition, protein solubility has been a major difficulty encountered (Chapman and Barratt, 1997). The rhZP3 expressed in CHO cells has been shown to possess acrosome reaction-inducing activity. However, no data are available regarding sperm binding in validated assays (van Duin et al, 1994; Barratt and Hornby, 1995). The fact that such recombinant proteins lack full sperm binding activity points to inadequate glycosylation of the protein core by the host cells.
In our studies we have cloned and expressed the cDNA of human ZP3 by stable transfection in a human ovarian cell line (PA-1 cells). This cell line was chosen to fit the glycosylation criterion, since glycosylation is tissue- and species-specific (Varki, 1993). The PA-1 cells produce glycosylated proteins such as lactosaminoglycan-carrier glycoprotein (Fukuda et al, 1985), heparin-binding protein (Furukawa et al, 1990) and fibronectin (Mcllhinney and Patel, 1983), and have been successfully used as an expression host to express other glycoproteins such as Interleukin-6 receptor (Lust et al, 1995). We purified the recombinant glycoprotein product and characterized its biological activities as sperm ligand (in competition studies using a homologous sperm-zona pellucida binding bioassay) and as physiologic inducer of the acrosome reaction (triggering exocytosis of sperm in suspension and assessing the frequency of acrosome reaction by lectin binding fluorescence). A first description of the full biological activities of this product has been reported (Dong et al, 2000). Here we have focused in the molecular biology and biochemical steps involved in cloning and expression as well as in glycoprotein purification.
The zona pellucida protein 3 (ZP3) is an essential component of the reproductive system as it functions as sperm receptor on the zona and as trigger of the acrosome reaction. To date, no recombinant human ZP3 (rhZP3) with well-documented and characterized biological activity is available. The aim of these studies was to clone and express a biologically active rhZP3 in human ovarian cells. A full-length human ZP3 cDNA was generated by RT-PCR using mRNA isolated from a human ovary. Sequencing of both strands demonstrated identical composition to the previously published cDNA sequence. An in vitro transcription and translation system revealed a protein core of 47 Kd for the product. To express the human ZP3 in vitro, the ZP3 cDNA with a six-histidine tail in its 3′ end was inserted into a pcDNA vector with a CMV promoter. The expression construct was introduced into PA-1 cells by stable transfection. The purification of rhZP3 was performed using Wheat Germ Agglutinin, DEAE ion exchange and Ni-NTA affinity chromatography. Western blot analysis confirmed a molecular weight of approximately 65 Kd for the secreted glycoprotein which had a PI of 4.60±0.05. Glycosylation labeling experiments demonstrated incorporation of 3H-galactose by the transfected cells. The rhZP3 demonstrated specific activities as ligand and inducer of the acrosome reaction of live human sperm.